Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Abstract

The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl-and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.Enzymes in organic solvents, instead of their natural aqueous milieu, remain synthetically useful catalysts (1-6). In addition, they display remarkable properties, e.g., solvent-dependent selectivity (7,8). Such solvent dependences of prochiral selectivity (9) and enantioselectivity (10) of crystalline enzymes in nonaqueous media have been nearly quantitatively predicted using structure-based molecular modeling and thermodynamic calculations.Despite recent advances (11)(12)(13)(14), the question of why enzymatic activity is often much reduced in organic solvents compared with water is far from resolved. Such an understanding, like that concerning the selectivity, requires both structural and mechanistic information. Recently, structures of several enzymes in neat organic solvents, namely those of subtilisin Carlsberg in acetonitrile (15, 16) and dioxane (17), ␥-chymotrypsin in hexane (18,19), and elastase in acetonitrile (20), have been elucidated and found to be essentially the same as in water. Furthermore, the structures in hexane of a ␥-chymotrypsin-product complex has been determined (19) and that of a tetrahedral complex claimed (18). However, to provide further insight into the enzyme mechanism in organic solvents, structures of covalent reaction intermediates should be determined. To the best of our knowledge, no such structures have been available, until now.All the aforementioned enzyme structures are of serine proteases, for which the universal reaction intermediate in water is an acyl-enzyme. In organic solvents, however, the formation of such an intermediate in the catalysis by subtilisin is supported by kinetic but not direct structural data (21-24).Moreover, it is simply presumed that the structure of the enzyme-substrate intermediate formed in different solvents is the same (9, 10). The most direct way to experimentally test this assumpti...

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